The purpose of the proposed research is uncover the genetic changes that occur when one animal species becomes two. Specifically, this research will identify and characterize genes or gene regions that cause behavioral mating discrimination in Drosophila pseudoobscura. Unlike genes isolated to date, the particular genes to be identified in the proposed work reduce interbreeding in the wild in the present day, and therefore have contributed to speciation directly. This species is ideal for the proposed research because of the availability of the full genome sequence and the extensive previous research performed on the genetics of behavioral mating discrimination and speciation in its group. Three goals are proposed: 1) precisely define the gene(s) or regions involved in discrimination via introgression, and conduct pilot studies to potentially confirm the candidate gene identities using transgenic techniques, 2) assess the frequency of the high discrimination alleles within D. pseudoobscura at the identified loci, and 3) resequence the regions bearing the loci involved in discrimination in multiple strains both to identify candidate genes or SNPs by association and to examine patterns of natural selection that may have operated on these regions. Several subsidiary goals are also described. This research will fill an important conceptual void and may aid in the understanding of the origin of species in other animal groups, including humans. It will also advance the area of behavioral genetics and the genetics of complex traits using a model system, with potential implications for developing hypotheses regarding human behaviors and traits.